Fluorescent versions of TCR and co-receptor proteins will be used to analyze formation of the immunological synapse in living tissue in a model of tolerance or diabetes induction. This project is to develop methods and techniques to allow analysis of cell surface molecular movement and intermolecular interaction in situ. Transgenic mice will be made expressing the fluorescent constructs [CD3zeta and CD8 fused to cyan and yellow fluorescent proteins (CFP, YFP, respectively)]. These will be crossed to a TCR transgenic mouse line that can cause diabetes or be rendered tolerant under different conditions. Using fluorescent deconvolution microscopy, fluorescence resonance energy transfer (FRET), and two-photon microscopy, the activities of these cells, and the TCR and coreceptor movements and interactions during tolerance or diabetes induction will be investigated. Cytotoxic T lymphocytes (CTLs) transduced with lentivirus vectors containing the fluorescent protein (FP)-chimeric genes will be used to set up the conditions for imaging tissue slices. Cells will be injected into sublethally irradiated mice transgenic for hemagglutinin (HA) expressed in the pancreas (Ins-HA mice) or control mice in an experiment expected to induce diabetogenesis. Pancreatic lymph nodes and pancreas will be used to test methods for in situ imaging of the fluorescent T cells in living tissue slices. Comparisons will be made to standard histology and immunohistochemistry to determine the best methods for analyzing live cells and to help in interpretation of morphology seen with this technique. Bone marrow chimeras (and/or fetal thymus organ culture and thymus reaggregation culture) will be used for analysis of tolerance induction in the living thymus tissue. Cells in a normal (positive selecting) environment will be compared with those in tolerogenic thymus. Acute (peptide-induced) and "genetic" tolerance induction using the appropriate major histocompatibility complex will be compared, as will differences between cortex and medulla. Conditions where naive T cells are tolerized in peripheral (pancreatic) lymph nodes or activated for diabetes will be analyzed.